Tailored liposomes for the treatment of bacterial infections

ABSTRACT

The invention relates to the use of empty liposomes of defined lipid composition or mixtures of empty liposomes of defined lipid composition and to the use of other lipid bilayers or monolayers of defined lipid composition for the treatment and prevention of bacterial infections. It has been found that such liposomes, in particular a two-and a four-component mixture of liposomes comprising cholesterol and sphingomyelin, liposomes consisting of sphingomyelin, liposomes comprising sphingomyelin and phosphatidylcholine, and liposomes comprising cholesterol and phosphatidylcholine efficiently sequestrate a variety of toxins secreted by bacteria, thus preventing binding of bacterial toxins to target cells and toxin-induced lysis of the target cells. Injected intravenously, liposome mixtures prevented death of laboratory mice infected with lethal doses of Staphylococcus aureus or Streptococcus pneumoniae.

FIELD OF THE INVENTION

The invention relates to the use of empty liposomes or liposome mixturesand to the use of other lipid bilayers or monolayers of defined lipidcomposition for the treatment and prevention of bacterial infections.Likewise the invention relates to a treatment of such bacterialinfections comprising administering empty liposomes or liposomemixtures, alone or in combination with standard antibiotic treatment.Furthermore the invention relates to new liposome mixtures as such.

BACKGROUND OF THE INVENTION

Bacterial infections remain one of the major threats to human lives. Asbacterial resistance to even the most potent antibiotics increases, sotoo must the efforts to identify novel anti-bacterial strategies. Amongother virulence factors, many pathogenic bacteria secrete toxins thatkill eukaryotic cells by disturbing their plasma membrane. Bacterialpore-forming toxins are active on the cell surface, causing poreformation and disruption of the plasma membrane followed by either lysisor apoptosis of host target cells, whereas bacterial phospholipasesinduce the death of host cells by enzymatic degradation of plasmalemmalphospholipids.

Bacterial membrane-destabilizing toxins, such as cholesterol-dependentcytolysins (CDCs: pneumolysin O, streptolysin O, tetanolysin),α-hemolysin or bacterial phospholipases (phospholipase C,sphingomyelinase) play a critical role in the establishment andprogression of infectious diseases. Such diseases are pneumonia, a majorcause of death among all age groups and the leading cause of death inchildren in low income countries; bacteremia, a severe complication ofinfections or surgery, which is characterized by high mortality due tosepsis and septic shock; and meningitis, a life-threatening disease,which also leads to serious long-term consequences such as deafness,epilepsy, hydrocephalus and cognitive deficits.

To target host cells bacterial membrane-destabilizing toxins either bindto individual membrane lipids (lipid head groups) or exploit thenon-homogenous nature of the lipid bilayer of eukaryotic cells' plasmamembrane, interacting with microdomains enriched in certain lipidspecies (Gonzales M. R. et al., Cell. Mol. Life Sci. 2008, 65:493-507).The non-homogenous distribution of lipids within the bilayer is notfavored by in vivo conditions since transmembrane proteins and thepresence of a multitude of individual lipid species with variablelengths and saturation status of their acyl chain oppose lipid de-mixingand thus the formation of stable lipid microdomains (Simons K. and GerlM. J., Nat. Rev. Mol. Cell Biol. 2010, 11:688-99). However, lipidde-mixing can be taken to its extremes in artificial protein-freeliposomes, manufactured from a limited number of carefully selectedlipid species, where extended, stable lipid microdomains can be created(Klose C. et al., J. Biol. Chem. 2010, 285:30224-32). Moreover,artificial liposomes allow for much higher relative concentrations of aparticular lipid than those ever likely to occur in vivo. Therefore,liposomes displaying stable lipid microdomains of defined biochemicalproperties and possessing high relative concentrations of particularlipids can be produced. Liposomes are currently used in the cosmetic andpharmaceutical industries as carriers for topical and systemic drugdelivery and are considered to be non-toxic.

SUMMARY OF THE INVENTION

The invention relates to the use of empty liposomes of defined lipidcomposition or mixtures of empty liposomes of defined lipid compositionfor the treatment and prevention of bacterial infections, in particularskin lesions, bacteremia, meningitis, respiratory tract infections, suchas pneumonia, and abdominal infections, such as peritonitis.

The invention furthermore relates to lipid bilayers or lipid monolayersof defined lipid composition covering non-lipid surfaces, for use in thetreatment and prevention of bacterial infections.

Likewise the invention relates to a treatment of such bacterialinfections comprising administering to a patient in need thereof atherapeutically effective amount of empty liposomes of defined lipidcomposition or mixtures of empty liposomes of defined lipid composition,and to a method of prevention of such bacterial inventions in a subjectat risk. Furthermore the invention relates to a treatment of bacterialinfections comprising administering to a patient in need thereof atherapeutically effective amount of empty liposomes of defined lipidcomposition or mixtures of empty liposomes of defined lipid composition,before, after, together or in parallel with a standard antibiotictreatment of the bacterial infection.

Furthermore the invention relates to new mixtures of empty liposomes ofdefined lipid composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Liposomes composed of cholesterol and sphingomyelin protectmonocytes from cholesterol-dependent cytolysins, α-hemolysin andphospholipase C.

A-D) Liposomes (1:1 w/w mixtures) containing cholesterol in combinationwith PC (3), Sm (4) or PS (5) but not with PE (6) protected THP-1 cellsfrom cholesterol-dependent cytolysins: pneumolysin 0 (A), streptolysin 0(B), tetanolysin (C) as well as from phospholipase C (D).

E) Liposomes (1:1 w/w) containing cholesterol in combination with Sm (4)but not with PC (3), PS (5) or PE (6) protected THP-1 cells fromα-hemolysin.

F) Liposomes without cholesterol (7-9) were ineffective.

c (r.u.)=number of cells, maintained in the presence of a toxin (1, 3-9)related to the number of cells maintained in the absence of a toxin (2),is given in relative units (r.u.). PLY=pneumolysin O; SLO=streptolysinO; TL=tetanolysin; PLC=phospholipase C; HML=α-hemolysin. 1=Control (noliposomes); 2=Control (no toxin), 3=Ch:PC (1:1 w/w) liposomes; 4=Ch:Sm(1:1 w/w) liposomes; 5=Ch:PS (1:1 w/w) liposomes; 6=Ch:PE (1:1 w/w)liposomes; 7=PC:Sm (1:1 w/w) liposomes; 8=Sm liposomes; 9=PC liposomes.Ch=cholesterol; PC=phosphatidylcholine; PS=phosphatidylserine;Sm=sphingomyelin; PE=phosphatidylethanolamine.

FIG. 2. Liposomes composed of cholesterol and sphingomyelin (1:1 w/w)protect monocytes from cholesterol-dependent cytolysins at microgramamounts, whereas 25-100 micrograms of the liposomes is required forprotection against Staphylococcus aureus α-hemolysin and Clostridiumperfringens phospholipase C.

A) Protection against 0.2 microgram of PLY. B) Protection against 0.4microgram of SLO. C) Protection against 0.2 microgram of TL. D)Protection against 1.2 microgram of HML. E) Protection against 4.5microgram of PLC.

c (r.u.)=number of cells, maintained in the presence of a toxin relatedto the number of cells maintained in the absence of a toxin, given inrelative units. X-axis: LP (mkg)=amount of liposomes in micrograms.PLY=pneumolysin O; SLO=streptolysin O; TL=tetanolysin; HML=α-hemolysin;PLC=phospholipase C.

FIG. 3. Cholesterol in concentrations above 30% (w/w) is required forliposomes composed of cholesterol and sphingomyelin to protect monocytesfrom pneumolysin, tetanolysin or α-hemolysin.

A) Protection against 0.2 microgram of PLY. B) Protection against 0.2microgram of TL. C) Protection against 1.2 microgram of HML. c(r.u.)=number of cells, maintained in the presence of a toxin related tothe number of cells maintained in the absence of a toxin, given inrelative units. Ch (%)=percentage of cholesterol (w/w) in liposomescomposed of cholesterol and sphingomyelin. PLY=pneumolysin;TL=tetanolysin; HML=α-hemolysin.

FIG. 4. Liposomes composed of cholesterol and sphingomyelin protectmonocytes from a combination of cholesterol-dependent cytolysins and S.aureus α-hemolysin.

A) Ch:Sm (1:1 w/w) liposomes (6) exerted fully protective effectsagainst the combined action of α-hemolysin (HML; 1.2 microgram),streptolysin O (SLO; 0.4 microgram) and tetanolysin (TL; 0.2 microgram),whereas Ch:PC (1:1 w/w) liposomes were ineffective (7). c (r.u.)=numberof cells, maintained in the presence of toxins (2-7) related to thenumber of control cells maintained in the absence of toxins (1), inrelative units. 1=Control (no toxins); 2=SLO, no liposomes; 3=TL, noliposomes; 4=HML, no liposomes; 5=SLO+TL+HML, no liposomes;6=SLO+TL+HML, Ch:Sm liposomes; 7=SLO+TL+HML, Ch:PC liposomes.Ch=cholesterol; PC=phosphatidylcholine; Sm=sphingomyelin. B) The fullprotective effect against the combined action of HML (1.2 microgram),SLO (0.4 microgram) and TL (0.2 microgram) was observed at 25 microgramof liposomes composed of Ch:Sm (1:1 w/w). LP (mkg)=amount of liposomesin micrograms. C) Centrifugation experiments confirmed that all threetoxins bind directly to Ch:Sm (1:1 w/w) liposomes. The toxins werepre-incubated with (6) or without (2-5) Ch:Sm liposomes. Aftercentrifugation, the supernatants were added to the cells. 1=Control (notoxins); 2=SLO, no liposomes; 3=TL, no liposomes; 4=HML, no liposomes;5=SLO+TL+HML, no liposomes; 6=SLO+TL+HML, Ch:Sm liposomes.

FIG. 5. Liposomes composed of cholesterol and sphingomyelin (1:1 w/w)protect monocytes from Streptococcus pyogenes toxins.

A,B) THP-1 cells proliferate in the presence of BHI broth (squares;dashed line in (A)). Culture supernatants of 5 Streptococcus pyogenesstrains (GAS 1-5=clinical isolates; all grown in BHI broth) effectivelykilled THP-1 cells (triangles), however the cells were protected fromthe effect of streptococcal toxins by liposomes composed of cholesteroland sphingomyelin (circles). 10⁵c=number of cells×10⁵. t(d)=time aftertreatment (days).

C) Liposomes composed of cholesterol and sphingomyelin protect monocytesfrom culture supernatants of Streptococcus pyogenes in microgramamounts. c(%)=percentage of cells, maintained in the presence ofbacterial supernatants related to cells maintained in the absence of thebacterial supernatants (100%). LP (mkg)=amount of liposomes inmicrograms.

FIG. 6. Liposomes composed of cholesterol and sphingomyelin (1:1 w/w) incombination with sphingomyelin-only liposomes completely protectmonocytes from Streptococcus pneumoniae toxins.

A,B) THP-1 cells proliferate in the presence of BHI broth (squares).Culture supernatants of 2 Streptococcus pneumoniae strains (Pneumo1=clinical isolate and Pneumo 2=D39 strain; both grown in BHI broth)effectively killed THP-1 cells (triangles), however the cells werepartially protected from the effect of Streptococcus pneumoniae toxinsby liposomes composed of cholesterol and sphingomyelin (1:1 w/w)(circles). 10⁵c=number of cells×10⁵. t(d)=time after treatment (days).

C) The mixture of cholesterol-containing and cholesterol-free,sphingomyelin-only liposomes was fully protective against Streptococcuspneumoniae toxins. The graph shows the protective effect of theliposomal mixtures composed of constant (400 μg) amount ofcholesterol:sphingomyelin (1:1 w/w) liposomes and varying amounts (0-400μg) of sphingomyelin-only liposomes.

c (r.u.)=number of cells, maintained in the presence of a bacterialsupernatant related to the number of cells maintained in the absence ofthe supernatant, is given in relative units (r.u.). Sm LP (mkg)=amountsof sphingomyelin-only liposomes in micrograms.

FIG. 7. Cholesterol:phosphatidylcholine liposomes (1:1 w/w) and amixture of cholesterol: phosphatidylcholine (1:1 w/w) and sphingomyelinliposomes protect monocytes from toxins secreted by Staphylococcusaureus strain MRSA 2040.

THP-1 cells proliferate in the presence of BHI broth (squares). Culturesupernatants of Staphylococcus aureus (grown in BHI broth) effectivelykill THP-1 cells in the absence of liposomes (triangles). (A) 900microgram (diamonds) of cholesterol:phosphatidylcholine (1:1 w/w)liposomes provide significant protection against bacterial toxins,whereas only limited protection was observed at 600 microgram (circles).(B) The full protection was observed for a mixture of 600 microgram ofcholesterol:phosphatidylcholine (1:1 w/w) liposomes with 75 microgram ofSm liposomes (diamonds). 900 microgram of Sm liposomes used alone wasineffective (circles). 10⁵c=number of cells×10⁵. t (d)=time aftertreatment (days).

FIG. 8. Cholesterol-free, sphingomyelin-containing liposomes and amixture of cholesterol:phosphatidylcholine (1:1 w/w) andsphingomyelin-only liposomes protect monocytes from toxins secreted byStaphylococcus aureus Doppelhof strain.

A,B) THP-1 cells proliferate in the presence of BHI broth (squares).Culture supernatants of Staphylococcus aureus (grown in BHI broth)effectively kill THP-1 cells in the absence of liposomes (triangles).(A) 1200 microgram (diamonds) of sphingomyelin liposomes providedsignificant protection against bacterial toxins. (B) When used at 600microgram, the most potent protection was observed for a mixture ofsphingomyelin and sphingomyelin:phosphatidylcholine (1:1 w/w)(diamonds), whereas sphingomyelin liposomes alone (circles) andsphingomyelin:phosphatidylcholine (1:1 w/w) liposomes alone (asterisks)were less effective. C) A mixture of cholesterol-containing andcholesterol-free, sphingomyelin-only liposomes was fully protectiveagainst toxins secreted by the Staphylococcus aureus Doppelhof strain.The graph shows the protective effect of the liposomal mixtures composedof constant (600 μg) amount of cholesterol:phosphatidylcholine (1:1 w/w)liposomes and varying amounts (0-1200 μg) of sphingomyelin-onlyliposomes.

10⁵c=number of cells×10⁵. t (d)=time after treatment (days). c(r.u.)=number of cells, maintained in the presence of a bacterialsupernatant related to the number of cells maintained in the absence ofthe supernatant, is given in relative units (r.u.). Sm LP (mkg)=amountsof sphingomyelin-only liposomes in micrograms.

FIG. 9. A 4-component mixture of cholesterol:sphingomyelin (1:1 w/w),sphingomyelin-only, sphingomyelin:phosphatidylcholine (1:1 w/w) andcholesterol:phosphatidylcholine (1:1 wt/wt) liposomes protect monocytesfrom toxins secreted by both Doppelhof and MRSA 2040 strains ofStaphylococcus aureus.

THP-1 cells proliferate in the presence of BHI broth (squares). Culturesupernatants of MRSA 2040 (A) or Doppelhof (B) Staphylococcus aureusstrains (grown in BHI broth) effectively kill THP-1 cells in the absenceof liposomes (triangles), however the cells are completely protectedfrom either MRSA 2040 (A) or Doppelhof (B) toxins by 1200 microgram ofthe 4-component liposomal mixture (1:1:1:1).

10⁵c=number of cells×10⁵. t (d)=time after treatment (days).

FIG. 10. Liposomes protect mice from Staphylococcus aureus bacteremia,from Streptococcus pneumoniae pneumonia and from Streptococcuspneumoniae bacteremia.

A) Laboratory mice were injected intravenously with a lethal dose of theDoppelhof Staphylococcus aureus strain. At 1, 5 and 24 hours afterinjection of bacteria, the mice were injected with either 25-50microliter of normal saline (diamonds) or 25 microliter (1 mg) ofcholesterol:sphingomyelin (1:1 w/w) liposomes (squares) or 50 microliter(2 mg) of a 1:2:2 mixture of cholesterol:sphingomyelin (1:1 w/w)liposomes+sphingomyelin-only liposomes+sphingomyelin:phosphatidylcholine(3:1 w/w) liposomes (triangles).

B) Mice were infected intranasally with the Streptococcus pneumoniaestrain D39. 30 minutes following injection of bacteria, the micereceived either an injection of 50 microliter of normal saline(diamonds) or a single intranasal injection of 50 microliter (2 mg) of a1:1:1:1 mixture of cholesterol:sphingomyelin (1:1w/w)+cholesterol:phosphatidylcholine (1:1w/w)+sphingomyelin-only+sphingomyelin:phosphatidylcholine (3:1 w/w)liposomes (triangles).

C) Mice were injected intravenously with a lethal dose of the S.pneumoniae strain D39. At 8 and 12 hours following injection ofbacteria, the mice received intravenously 75 microliter/injection (3 mg)of the following liposomes: 1) a 1:1 mixture ofcholesterol:sphingomyelin (1:1 w/w)+sphingomyelin-only liposomes(triangles); 2) cholesterol:sphingomyelin (1:1 w/w) liposomes (squares);3) sphingomyelin-only liposomes (circles) or 4) normal saline(diamonds).

S (%)=percent surviving mice. t (d)=time after infection (days)

DETAILED DESCRIPTION OF THE INVENTION

Engineered to possess higher than in vivo affinities formembrane-targeting toxins, inhaled or intravenously injected or infusedempty liposomes and liposome mixtures serve as traps for bacterialtoxins residing in blood or airways of infected patients, paving a wayfor a novel anti-bacterial toxin-sequestrating therapy.

The invention relates to the use of empty liposomes of defined lipidcomposition or mixtures of empty liposomes of defined lipid compositionfor the treatment and prevention of bacterial infections, in particularbacteremia, meningitis, bacterial skin infections, respiratory tractinfections, such as pneumonia, and abdominal infections, such asperitonitis.

Liposomes of the invention are empty liposomes, i.e. liposomes notencapsulating any antibiotic or other drug. They may, if desired, beused in combination with known or novel liposomes carrying drugs.

The present study shows that artificial liposomes of precisely definedlipid composition or mixtures of liposomes of precisely defined lipidcomposition efficiently sequestrate purified pore-forming toxins andphospholipase C, thereby preventing their binding to the target cells.Consequently, the application of liposomes or their mixtures prevent thelysis of cultured epithelial cells and monocytes induced by theapplication of purified toxins or culture supernatants of Streptococcuspneumonia, Streptococcus pyogenes and Staphylococcus aureus and protectlaboratory mice from death due to an experimentally induced bacteremiaor pneumonia.

The invention relates to the use of empty liposomes of defined lipidcomposition or mixtures of empty liposomes of defined lipid compositionfor the treatment and prevention of bacterial infections.

The invention furthermore relates to lipid bilayers or lipid monolayersof defined lipid composition covering non-lipid surfaces, for use in thetreatment and prevention of bacterial infections. Non-lipid surfacesconsidered are, for example, medical appliances, biodegradable beads,and nanoparticles.

Liposomes considered are artificial liposomes of 20 nm to 10 μm,preferably 20 to 500 nm, comprising lipids or phospholipids selectedfrom the group of sterols, sphingolipids and glycerolipids, inparticular selected from the group consisting of cholesterol,sphingomyelins, ceramides, phosphatidylcholines,phosphatidylethanolamines, phosphatidylserines, diacylglycerols, andphosphatidic acids containing one (lyso-) or two (diacyl-), saturated orunsaturated fatty acids longer than 4 carbon atoms and up to 28 carbonatoms.

The composition of lipid bilayers or lipid monolayers considered is thesame as indicated for liposomes.

Fatty acids comprising between 4 and 28 carbon atoms are, for example,saturated linear alkanecarboxylic acids, preferably with an even numberof carbon atoms, such as between 12 and 26 carbon atoms, for examplelauric, myristic, palmitic, stearic, arachidic, or behenic acid, orunsaturated linear alkenecarboxylic acids, preferably with an evennumber of between 12 and 26 carbon atoms and one, two or more,preferably up to six double bonds in trans or, preferably cisconfiguration, for example oleic acid, linoleic acid, alpha-linoleicacid, arachidonic acid, or erucic acid.

Empty liposomes means that the liposomes considered in the presentinvention do not incorporate antibiotic or other drugs. “Incorporated”as used herein means encapsulated into the cavity of the liposome,within the potential double layer of the liposome, or as part of themembrane layer of the liposome. Liposomes as used herein also excludeliposomes modified with binding agents such as antibodies and mono- oroligosaccharides, e.g. as in glycolipids. However, liposomes modifiedwith polyethylene glycol (PEG) are considered as part of this invention.PEG is known to modify the circulation time of liposome.

In particular, the invention relates to the use of empty liposomescomprising cholesterol and sphingomyelin, and of mixtures of emptyliposomes comprising cholesterol and sphingomyelin, orphosphatidylcholine and sphingomyelin, with other empty liposomes ofdefined lipid composition, such as liposomes comprising lipids orphospholipids selected from the group consisting of sterols,sphingolipids and glycerolipids, in particular selected from the groupconsisting of cholesterol, sphingomyelins, ceramides,phosphatidyl-cholines, phosphatidylethanolamines, phosphatidylserines,diacylglycerols, and phosphatidic acids containing one or two saturatedor unsaturated fatty acids longer than 4 carbon atoms and up to 28carbon atoms, for the treatment and prevention of bacterial infections.

In one embodiment, the invention relates to the use of empty liposomescomprising sphingomyelin and 30% (w/w) or more cholesterol, and ofmixtures of empty liposomes comprising sphingomyelin and 30% (w/w) ormore cholesterol with other empty liposomes as defined herein, for thetreatment and prevention of bacterial infections. In particular, theinvention relates to the use of empty liposomes consisting ofsphingomyelin and of 30% (w/w) or more cholesterol, and of mixtures ofempty liposomes consisting of sphingomyelin and of 30% (w/w) or morecholesterol with other empty liposomes as defined herein, for thetreatment and prevention of bacterial infections. More particularly, theinvention relates to the use of empty liposomes consisting ofsphingomyelin and of between 35% and 65% (w/w) cholesterol, preferablybetween 40% and 55% (w/w) cholesterol, in particular between 45% and 55%(w/w) cholesterol, such as around 50% (w/w) cholesterol, and of mixturesof empty liposomes consisting of sphingomyelin and of between 35% and65%, or 40% and 55%, or 45% and 55%, e.g. around 50% (w/w) cholesterolwith other empty liposomes as defined herein, for the treatment andprevention of bacterial infections.

In a particular embodiment, the invention relates to the use of aliposome mixture of empty liposomes comprising or consisting ofcholesterol and sphingomyelin, with other empty liposomes comprising orconsisting of sphingomyelin, for the treatment and prevention ofbacterial infections.

In another embodiment, the invention relates to the use of a liposomemixture of empty liposomes comprising or consisting ofphosphatidylcholine and sphingomyelin with other empty liposomes asdefined herein, for the treatment and prevention of bacterialinfections. In particular, the invention relates to the use of aliposome mixture of empty liposomes comprising or consisting ofphosphatidylcholine and sphingomyelin with other empty liposomescomprising or consisting of sphingomyelin, for the treatment andprevention of bacterial infections.

In a particular embodiment, the invention relates to the use of athree-component liposome mixture of empty liposomes comprising orconsisting of cholesterol and sphingomyelin with other empty liposomescomprising or consisting of phosphatidylcholine and sphingomyelin, andwith empty liposomes consisting of sphingomyelin, for the treatment andprevention of bacterial infections.

In yet another particular embodiment, the invention relates to the useof a four-component liposome mixture of empty liposomes comprising orconsisting of cholesterol and sphingomyelin with other empty liposomescomprising or consisting of phosphatidylcholine and sphingomyelin, withempty liposomes consisting of sphingomyelin, and with empty liposomescomprising or consisting of cholesterol and phosphatidylcholine, for thetreatment and prevention of bacterial infections.

The particular composition of lipid bilayers or lipid monolayersconsidered is the same as indicated for liposomes, preferably comprisingcholesterol and sphingomyelin, and optionally phosphatidylcholine.

It is understood that the empty liposomes as defined above, the mixturesof empty liposomes as defined above, and the lipid bilayers or lipidmonolayers may be used together with further compounds. For example, itis possible to add components to prepare standard pharmaceuticalcompositions. It is also considered to add drugs or drug-like compounds,or to add further liposomes incorporating drugs or drug-like compoundsin the liposome interior.

Drugs considered are, in particular, antibiotics. Such antibiotics are,for example, carbapenems, such as imipenem/cilastatin, meropenem,ertapenem, and doripenem; 1^(st) generation cephalosporins, such ascefadroxil and cefalexin; 2^(nd) generation cephalosporins, such ascefuroxime, cefaclor, and cefprozil; 3^(rd) generation cephalosporins,such as ceftazidime, ceftriaxone, cefixime, cefdinir, cefditoren,cefotaxime, cefpodoxime, and ceftibuten, 4^(th) generationcephalosporins, such as cefepime; 5^(th) generation cephalosporins, suchas ceftaroline fosamil and ceftobiprole; glycopeptides, such asvancomycin, teicoplanin, and telavancin; macrolides, such asclarithromycin, azithromycin, dirithromycin, erythromycin,roxithromycin, troleandomycin, telithromycin, spectinomycin, andspiramycin; penicillins, such as amoxicillin, flucloxacillin, oxacillin,carbenicllin, and piperacillin; penicillin combinations, such asamoxicillin/clavulanate, piperacillin/tazobactam, ampicillin/sulbactam,and ticarcillin/clavulanate; quinolones, such as ciprofloxacin (e.g.Aradigm's liposomal ciprofloxacin) and moxifloxacin; drugs againstmycobacteria, such as rifampicin (rifampin in US), clofazimine, dapsone,capreomycin, cycloserine, ethambutol, ethionamide, isoniazid,pyrazinamide, rifabutin, rifapentine, and streptomycin; otherantibiotics, such as metronidazole, arsphenamine, chloramphenicol,fosfomycin, fusidic acid, linezolid, mupirocin, platensimycin,quinupristin/dalfopristin, rifaximin, thiamphenicol, tigecycline, andtinidazole; aminoglycosides, such as amikacin, gentamicin, kanamycin,neomycin, netilmicin, tobramycin (e.g. Axentis' fluidosomes™ tobramycin)and paromomycin; sulfonamides, such as mafenide, sulfonamidochrysoidine,sulfacetamide, sulfadiazine, silver sulfadiazine, sulfamethizole,sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole,trimethoprim, and trimethoprim-sulfamethoxazole (co-trimoxazole,TMP-SMX); tetracyclines, such as demeclocycline, doxycycline,minocycline, oxytetracycline, and tetracycline; lincosamides, such asclindamycin, and lincomycin; and lipopeptides, such as daptomycin.

Further drugs considered are anti-cancer agents, for example vincristinesulfate, vincristine, cytarabine, daunorubicin, and doxorubicin.

Still other drugs considered are anti-inflammatory drugs, for examplecorticosteroids (glucocorticoids), such as hydrocortisone (cortisol),cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate (DOCA), aldosterone, budesonide, desonide,and fluocinonide; non-steroidal anti-inflammatory drugs, e.g.salicilates, such as aspirin (acetylsalicylic acid), diflunisal, andsalsalate; propoinic acid derivatives, such as ibuprofen, dexibuprofen,naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen,oxaprozin, and loxoprofen; acetic acid derivatives, such asindomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, andnabumetone; enolic acid (oxicam) derivatives, such as piroxicam,meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam; fenamic acidderivatives (fenamates), such as mefenamic acid, meclofenamic acid,flufenamic acid, and tolfenamic acid; selective COX-2 inhibitors(coxibs), such as Celecoxib; and others, such as licofelone.

Further drugs considered are vasopressors and vasoconstrictors, forexample vasopressin, oxymetazoline, phenylephrine, propylhexedrine,pseudoephedrine, epinephrine, norepinephrine, dopamine, andantihistamines.

Also considered are other type of drugs, for example paracetamol (painkiller), amphotericin B (against fungal infections), bupivacaine(post-surgical pain control), vaccines against hepatitis A, influenza,tetanus, evasive MRSA, pertussis, diphtheria, meningococcus, cholera,typhoid, anthrax, pneumococcus (e.g. Prevnar 13®), and otherantibacterial vaccines, morphine (pain killer), verteporfin(ophthalmological diseases), estradiol (menopausal disturbances),aganocide® compounds, e.g. auriclosene (NVC-422,N,N-dichloro-2,2-dimethyltaurine (anti-bacterials), M Bio Technology'sliposome particles comprising specific lipid bacterial antigens, e.g.bacterium mimic particles as vaccines (mycoplasma infections includingpneumonia), and bacteriophages.

Further drugs considered are antitoxins, for example tetanus antitoxin,such as tetanus immunoglobulin, nanosponges, polymeric nanoparticles,biomimetic polymeric nanoparticle core surrounded by host cell membranes(such as red blood cell membranes), toxin-targeting monoclonalantibodies and antibody fragments, natural compounds inhibiting specifictoxin productions, inhibitors of the bacterial toxin secretion system,such as T3SS inhibitors, toxin-binding mucin-type fusion proteins,soluble T cell receptors acting as decoy to neutralize toxins, andpeptides that inhibit the processing of toxins.

Furthermore the invention relates to new mixtures of empty liposomes ofdefined lipid composition. In particular, the invention relates tomixtures of empty liposomes comprising cholesterol and sphingomyelin, orphosphatidylcholine and sphingomyelin, with other empty liposomes ofdefined lipid composition, such as liposomes comprising lipids orphospholipids selected from the group of sterols, sphingolipids andglycerolipids, in particular selected from the group consisting ofcholesterol, sphingomyelins, ceramides, phosphatidylcholines,phosphatidylethanolamines, phosphatidylserines, diacylglycerols, andphosphatidic acids containing one or two saturated or unsaturated fattyacids longer than 4 carbon atoms and up to 28 carbon atoms, for thetreatment and prevention of bacterial infections.

Particular mixtures considered are mixtures of empty liposomescomprising or consisting of sphingomyelin and cholesterol with otherempty liposomes as defined herein, such as mixtures of empty liposomescomprising or consisting of cholesterol and sphingomyelin, with otherempty liposomes comprising or consisting of sphingomyelin.

Other particular mixtures considered are mixtures of empty liposomescomprising or consisting of phosphatidylcholine and sphingomyelin withother empty liposomes as defined herein, such as mixtures of emptyliposomes comprising or consisting of phosphatidylcholine andsphingomyelin with other empty liposomes comprising or consisting ofsphingomyelin.

Other particular mixtures considered are three-component liposomemixtures of empty liposomes comprising or consisting of cholesterol andsphingomyelin with other empty liposomes comprising or consisting ofphosphatidylcholine and sphingomyelin, and with empty liposomesconsisting of sphingomyelin.

Further particular mixtures considered are four-component liposomemixtures of empty liposomes comprising or consisting of cholesterol andsphingomyelin with other empty liposomes comprising or consisting ofphosphatidylcholine and sphingomyelin, with empty liposomes consistingof sphingomyelin, and with empty liposomes comprising or consisting ofcholesterol and phosphatidylcholine.

Within the liposomes, the components may be present in differentamounts, depending on the tendency to form liposomes, the stability ofthe liposomes of different composition, and the intended use. Examplesare liposomes consisting of two components in approximately 1:1, 2:1,3:1, 4:1, or 5:1 (weight per weight) composition. Further components maybe admixed in approximately 10, 20 or 25% (w/w) amounts.

In the preferred liposomes of the invention cholesterol is present in anamount of 30-70%, preferably 40-60%, e.g. 45-55%, in particular around50% (w/w), phosphatidylcholine is present in an amount of 10-60%,preferably 20-60%, preferably 40-60%, e.g. 45-55%, more preferablyaround 50% (w/w); and sphingomyelin is present in an amount of 10-100%,preferably 20-60% or 100%, preferably 40-60%, e.g. 45-55%, morepreferably around 50% (w/w) or 100%, the cholesterol:sphingomyelin ratiois between 5:1 and 1:2, preferably 2:1 and 1:2, in particular around 1:1(w/w), and the cholesterol: phosphatidylcholine orphosphatidylcholine:sphingomyelin ratio is between 5:1 and 1:5,preferably between 2:1 and 1:2, in particular around 1:1 (w/w).

In the liposomal mixtures individual liposome components with differentcomposition are mixed at proportions defined by treatment needs.Examples are approximately 1:1, 2:1, or 3:1 (w/w) for 2-componentmixtures; approximately 1:1:1, 2:1:1, or 2:2:1 (w/w) for 3-componentmixtures; and approximately 1:1:1:1, 2:1:1:1, 2:2:1:1, or 2:2:2:1 (w/w)for 4-component mixtures.

The liposomes considered consist of one or more phospholipid bilayers.Preferred are large unilamellar vesicles (LUVs) and multilamellarvesicles (MLVs). Most preferred are small unilamellar vesicles (SUVs).

The liposomes are manufactured according to extrusion or sonication ormicrofluidization (e.g. high pressure homogenization) methods known inthe art. For example, the lipids are mixed in an organic solvent such aschloroform. Chloroform is evaporated and the dry lipid film is hydratedin an aqueous solution such as normal saline (0.9% NaCl), Krebssolution, or Tyrode's solution and further sonicated to produceliposomes. If necessary, the size of the liposomes can be controlled bytheir extrusion through membrane filters of fixed pore diameter.Individually produced liposomes of different lipid compositions aremixed in the required proportions just before application.

Epithelial cells constitute the physical barrier to pathogens.Artificial liposomes are able to protect human embryonic kidney (HEK293) epithelial cells from streptolysin O (SLO) induced lysis. The SLOpores formed within the plasma membrane are large enough to cause anefflux of cytoplasmic proteins with M_(r) up to 100 kDa. The directbinding of SLO to cholesterol-containing liposomes was confirmed bypre-incubation of the liposomes with the toxin followed bycentrifugation. After centrifugation, liposomes, recovered in thepellet, were discarded and the liposome-free supernatants were added tothe cells. The supernatants did not inflict any damage on the exposedcells, suggesting that the toxin was efficiently removed from thesolution due to its binding to the liposomes.

Liposomes protected not only epithelial cells but also cells of theinnate immune system against a variety of pore-forming toxins (PFTs).The effects of PFTs on the proliferation of THP-1 human monocytic cellline were assessed in the presence or absence of liposomes of variouslipid compositions. Proliferation of THP-1 cells was completelyinhibited in the presence of 200 ng of pneumolysin (PLY), 400 ng ofstreptolysin O (SLO), 200 ng of tetanolysin (TL), 1.2 μg S. aureusα-hemolysin (HML), or 4.5 μg Clostridium perfringens phospholipase C. Asshown in FIG. 1A-D, liposomes containing cholesterol in combination (1:1w/w) with either phosphatidylcholine (PC), sphingomyelin (Sm) orphosphatidylserine (PS) but not with phosphatidylethanolamine (PE)protected THP-1 cells from cholesterol-dependent cytolysins (PLY, SLO,TL) or phospholipase C (PLC), whereas liposomes, which contained nocholesterol, were ineffective (FIG. 1F).

In contrast, Ch:PC liposomes (1:1 w/w) and Ch:PS liposomes (1:1 w/w)displayed no protective effect against S. aureus α-hemolysin, whichbelongs to the group of small pore forming toxins (FIG. 1E). Liposomes,which contained no cholesterol, were also ineffective (FIG. 1F).However, liposomes containing Ch:Sm (1:1 w/w) were able to exert a fullyprotective effect against α-hemolysin (FIG. 1E). Thus only liposomescomposed of cholesterol and sphingomyelin were able to protect THP-1cells from any of the tested toxins.

FIG. 2 shows that the full protective effect of Ch:Sm (1:1 w/w)liposomes against 200 ng PLY was observed at 3 μg; against 400 ng of SLOat 1.5 μg; against 200 ng TL at 3 μg; against 1.2 μg HML at 25-50 μg,and against 4.5 μg PLC at 100 μg.

FIG. 3 demonstrates that cholesterol in concentrations equal or above30% (w/w) was required for Ch:Sm liposomes to protect monocytes fromPLY, TL or HML. The maximal protection was observed at 50% (w/w) ofcholesterol which corresponds to 66 mol % of cholesterol.

Since liposomes composed of cholesterol and sphingomyelin were able toprotect cells from either cholesterol-dependent cytolysins or fromα-hemolysin, it was investigated whether these liposomes were effectiveagainst a combination of both toxin classes. Indeed, 25 μg of Ch:Sm (1:1w/w) liposomes exerted fully protective effects against the combinedaction of α-hemolysin (1.2 μg), SLO (400 ng) and TL (200 ng), whereasliposomes composed of Ch:PC (1:1 w/w) had no effect (FIG. 4A,B).Centrifugation experiments confirm that all three toxins bind directlyto Ch:Sm liposomes (FIG. 4C).

Ch:Sm liposomes are able to protect cultured cells from the entirepalette of toxins secreted by clinically relevant strains of bacterialpathogens. The proliferation of THP-1 cells was assessed in the presenceof the lytic concentrations of the bacterial culture supernatants and inthe presence or absence of liposomes.

As shown in FIG. 5A,B, Ch:Sm (1:1 w/w) liposomes protected the cellsfrom the effect of toxins secreted by Streptococcus pyogenes. The fullprotective effect against Streptococcus pyogenes toxin(s) was observedat microgram amounts of the liposomes (FIG. 5C). These amounts aresimilar to those required for neutralization of lytic concentrations ofpurified cholesterol-dependent cytolysins, but are much lower than thoserequired for the neutralization of either purified α-hemolysin orpurified phospholipase C (FIG. 2) suggesting that cholesterol-dependentcytolysins are solely responsible for the cytolytic action ofStreptococcus pyogenes.

Ch:Sm liposomes also protected the cells from the effect of toxinssecreted by Streptococcus pneumonia (FIG. 6A-C). Whereas only limitedprotection was achieved with cholesterol-containing (1:1 w/w) liposomes(FIG. 6A-C), the mixture of cholesterol-containing (400 μg) andcholesterol-free, Sm-only liposomes (400 μg) was fully protectiveagainst this pathogen (FIG. 6C).

Staphylococcus aureus is notorious for its resistance to the most potentantibiotics. The liposomal toxin-sequestration provides protection evenagainst this pathogen. Ch:Sm (1:1 w/w) liposomes showed only limitedprotection against toxins secreted by the methicillin-resistant strainof Staphylococcus aureus (MRSA 2040). Similar results were obtained forCh:PC (1:1 w/w) liposomes: as high as 900 μg of Ch:PC liposomes wasrequired to achieve significant protection, whereas 600 μg of theseliposomes showed only slight effect (FIG. 7A). However, the detailedanalysis of different liposomal mixtures demonstrated that addition ofas little as 75 μg of sphingomyelin-only liposomes to 600 μg of Ch:PC(1:1 w/w) liposomes achieved full protection against this pathogen (FIG.7B). Sm liposomes alone or PC liposomes alone had no protective effectat amounts as high as 900 μg (FIG. 7B).

The liposomal treatment was also efficient against a clinically relevant“Doppelhof” strain of Staphylococcus aureus, isolated from a septicpatient. Neither Ch:Sm (1:1 w/w) nor Ch:PC (1:1 w/w) liposomes nor theircombination with Sm-only liposomes were effective when used atconcentrations which were protective against MRSA 2040 strain. However,a detailed analysis of the protective action of various liposomalcompositions and their combinations demonstrated that—in contrast toMRSA 2040 strain—the cytolytic toxins secreted by the Doppelhof strainwere efficiently sequestrated by Sm-only liposomes (FIG. 8A). Whereas1200 μg of Sm liposomes alone showed significant protection against theDoppelhof strain, at lower concentrations the mixture containing Smliposomes and Sm:PC liposomes was more potent than the same amounts ofeither Sm liposomes or Sm:PC liposomes (FIG. 8B). The mixture ofcholesterol-containing (1:1 w/w; 600 μg) and cholesterol-free,sphingomyelin-only liposomes (1,200 μg) was fully protective againsttoxins secreted by the Staphylococcus aureus Doppelhof strain (FIG. 8C).

Thus, not only Staphylococcus aureus or Streptococcus pneumonia secretemultiple cytolytic toxins but also the relative amounts of secretedtoxins varies significantly between different strains necessitating theuse of complex liposomal mixtures to achieve high-affinity toxin bindingfor their full neutralization. However, due to non-ideal selectivity ofthe toxins-liposomes interactions, significant partial protection can bealready achieved with single liposomes, provided their concentration ishigh enough to promote their low-affinity toxin-binding.

Detailed analysis of different liposomal mixtures demonstrated that1,200 μg (total lipid) of a 1:1:1:1 mixture of Ch:Sm (1:1 w/w)liposomes+Ch:PC (1:1 w/w) liposomes+Sm-only+Sm:PC (1:1 w/w) liposomeswas required for protection against both MRSA 2040 and Doppelhof strainsof ‘Staphylococcus aureus (FIG. 9). Of importance, owing to the presenceof cholesterol-containing liposomes, the 4-component mixture alsoprotects against Streptococcal toxins (see FIGS. 1-5). Thus, thefour-component liposomal mixture (1200 μg of total lipid) is able toprotect cultured cells from a combined action of Streptococcal andStaphylococcal toxins. The two-component mixture consisting ofcholesterol-containing (50% w/w cholesterol) and sphingomyelin-onlyliposomes was also protective against all bacterial supernatants tested(FIGS. 6-8), however slightly higher amount (1′800 pg of total lipid) ofthis mixture was required for the full protection against toxinssecreted by the Staphylococcus aureus Doppelhof strain (FIG. 8 C).

The bacterial species tested (Streptococcus pneumoniae, Staphylococcusaureus and Streptococcus pyogenes) are known to induce or contribute tothe development of life-threatening conditions such as bacteremia. Thepreferred three- or four-component mixture of liposomes is able toprotect laboratory mice from experimentally induced bacteremia orpneumonia.

Mice were injected intravenously with a lethal dose of Staphylococcusaureus Doppelhof strain, a clinical isolate from a septic patient. At 1,5 and 24 hours following injection of bacteria, mice were injectedintravenously either with normal saline (control), with 1 mg/injectionof Ch:Sm (1:1 w/w) liposomes, or with 2 mg/injection of a 1:2:2 mixtureof Ch:Sm (1:1 w/w) liposomes; Sm-only and Sm:PC (1:1 w/w) liposomes. Nocontrol mice survived beyond day 7, with 90% of deaths occurring within36 hours (FIG. 10A). Mice treated with cholesterol-sphingomyelinliposomes survived 2-3 days longer than control ones but did not recoverafter bacteremia. However, treatment with the 3-component liposomalmixture resulted in complete recovery of 6 out of 8 mice.

In the pneumococcal pneumonia model, mice were infected intranasallywith the S. pneumoniae strain D39. 30 minutes following injection ofbacteria, the mice received a single intranasal injection of 2 mg of a1:1:1:1 mixture of Ch:Sm (1:1 w/w) liposomes+Ch:PC (1:1w/w)liposomes+Sm-only liposomes+Sm:PC (3:1 w/w) liposomes. FIG. 10B showsthat the liposomal mixture provided protection against pneumonia.

In the pneumococcal bacteremia model, mice were injected intravenouslywith a lethal dose of the S. pneumoniae strain D39. At 8 and 12 hoursfollowing injection of bacteria, the mice received intravenously 3mg/injection of the following liposomes: 1) a 1:1 mixture of Ch:Sm (1:1w/w) liposomes+Sm-only liposomes; 2) Ch:Sm (1:1 w/w) liposomes; 3)Sm-only liposomes or 4) normal saline. FIG. 10C shows that no control orSm-only mice survived beyond 32 hours. However, 6 out of 8 mice thatreceived Ch:Sm+Sm-only liposomal mixture and 3 out of 8 mice thatreceived Ch:Sm liposomes were still alive after 56 hours of bacteremia.

The doses of liposomes (50-150 mg/kg) required for the protection ofmice against Staphylococcal bacteremia are known to be non-toxic whenused as carriers for intravenous delivery of antibiotics in rats (400mg/kg; Bakker-Woudenberg I. A. J. M. et al., Antimicrobial Agents andChemotherapy, 2001, 45:1487-1492). Moreover, the recommended doses oflipid emulsions (e.g “Intralipid”, “Lipovenös”), which are infusedintravenously in patients suffering from dysfunctions in fatty acidmetabolism, contain, in addition to 2.7 g/kg of fatty acids,approximately 300 mg/kg of egg phospholipids; i.e. the phospholipidsused in the liposomal preparations of the present invention. Thus, it issafe to administer liposomes for the treatment of bacterial infectionsin human patients, and liposomes will not elicit adverse events.

The efficiency of liposomal toxin-sequestration can be further improved.Since the liposomes used in this study were mostly multilamellarliposomes and therefore at least half of their lipid content wasunavailable for toxin binding, the toxin-sequestrating capacity ofunilamellar liposomes is predicted to be at least twice as high.Liposomes composed of selected synthetic lipids, containing uniform acylchains, and additional lipid species (e.g. ceramide), known todramatically enhance bilayer lipid de-mixing, provide a better targetfor bacterial toxins than the liposomes manufactured from naturallipids, which were used in this study. Using PEG-derivatives ofphosphatidylethanolamine, the circulation time of liposomes and thustheir efficacy can be likewise significantly increased.

The lipid surface (bilayer) of liposomes forms spontaneously inwater-based solvents and therefore traps water and other water-solubleinorganic and organic molecules, which might be present during liposomeproduction, inside the liposome. The empty liposomes, used in thepresent study, are liposomes produced in buffers containing water andsimple organic or inorganic molecules (for example NaCl, KCl, MgCl₂,glucose, HEPES, and/or CaCl₂). However, complex organic molecules(antibiotics, vitamins, adjuvants, and others) can likewise be includedduring liposome production (loaded liposomes). These complex organicmolecules are not expected to interfere with the toxin-sequestratingproperties of the liposomes; however they will provide additionaltherapeutic effects.

The invention further relates to a treatment of bacterial infectionscomprising administering to a patient in need thereof a therapeuticallyeffective amount of empty liposomes of defined lipid composition ormixtures of empty liposomes of defined lipid composition, as describedhereinbefore.

Likewise the invention relates to the prevention of bacterial infectionscomprising administering to a subject exposed to the risk of infection apreventive amount of empty liposomes of defined lipid composition ormixtures of empty liposomes of defined lipid composition effective forprotection.

Bacterial infections considered are infections of the respiratory tract,gastrointestinal tract, urogenital tract, cardiovascular tract, or ofthe skin, as well as systemic infections caused by bacteria that producepore-forming toxins and phospholipases, for example caused by Aeromonashydrophila, Arcanobacterium pyogene, Bacillus thurgiensis, Bacillusanthracis, Bacillus cereus, Clostridium botulinum, Clostridiumperfringens, Clostridium septicum, Clostridium sordellii, Clostridiumtetani, Corynebacterium diphtheriae, Escherichia coli, Listeriamonocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus (includingMethicillin-resistant Staphylococcus aureus (MRSA)), Streptococcuspneumonia, Streptococcus pyogenes (also known as Group A Streptococcus(GAS)), Streptococcus equisimilis, Streptococcus agalactiae,Streptococcus suis, Streptococcus intermedius or Vibrio cholera.

Further bacterial infections considered are infections of thenasopharynx system, CNS system, meningeal membranes, vagina, bones (forexample osteomyelitis) and joints, kidney, skeletal muscles, outer ear(for example otitis externa), and eye, for example infectiousconjunctivitis, bacterial keratitis, and inner-eye infections.

Particular bacterial infections considered as target for a treatmentwith the liposomes as described above are bacteremia, bacteriallyinfected skin lesions, meningitis, respiratory tract infections, forexample pneumonia, and abdominal infections, such as peritonitis.

Dosages considered for the treatment or prevention of infections are 1mg to 300 g of liposomes (total lipid) per inhalation/injection/infusiononce or several times per day, preferably 100 mg to 10 g once to threetimes per day. A HED (human equivalent dose) is between 100 and 1000mg/m², preferably around 300 mg/m², or around 8 mg/kg in humans.

Liposomes can be administered as aerosol for the treatment ofrespiratory tract infections. The preparation of aerosols from liposomessuch as the empty liposomes of the invention is known in the art. Forexample the liquid suspension of liposomes can be delivered with ametered-dose inhaler (MDI), i.e. a device that delivers a specificamount of medication to the airways or lungs, in the form of a shortburst of aerosolized medicine that is inhaled by the patient.

For the treatment of bacterial infections of the skin, application ofthe liposomes of the invention is considered in the form of topicalpharmaceutical compositions, such as liquid suspensions and the like.The preparation of a suspension from liposomes such as the emptyliposomes of the invention is known in the art. For example, theliposome suspension prepared in normal saline or any other aqueoussolution can be applied directly to the skin.

For the treatment of systemic bacterial infections, empty liposomes ofthe invention are applied in the form of intravenous, intramuscular orsubcutaneous injections. Injection solutions are prepared by standardmethods known in the art, for example as suspensions of the liposomes insterile normal saline. Such suspensions can be directly injected. It isalso considered to apply the liposomes of the invention in a formulationuseful for sublingual or buccal application. For the treatment ofperitonitis, intraperitoneal application is considered. Eye drops may beused for bacterial infection of the eyes.

The empty liposomes of the invention will sequestrate bacterial toxinsand thus prevent bacteria from penetrating the host's epithelia or theirsystemic propagation. The development of systemic disease can thus beaverted or slowed; and the pathogens can be efficiently cleared by cellsof the host's innate immune system, which are likewise protected fromtoxins by the liposomes.

The liposomes themselves are not cytotoxic, nor are they bactericidal.Therefore, it is unlikely that they will exert selective antibacterialpressure, which would further the emergence of drug-resistant bacteria.The empty liposomes of the invention mimic structures that already existin the host's cells, in order to bait bacterial toxins. Therefore it isinconceivable that bacteria will adapt to the liposomal challenge: everyattempt to escape the bait by decreasing the affinity of their toxins tothe liposomes inevitably leads to the emergence of toxins which arelikewise ineffective against the host's cells.

Liposome-based chemotherapy is an appealing alternative to bothantibiotic therapy and to a treatment with toxin-sequestratingantibodies.

The invention also relates to a treatment of bacterial infectionscomprising administering to a patient in need thereof a therapeuticallyeffective amount of empty liposomes before, after, together or inparallel with a standard antibiotic treatment of the bacterialinfection. In combination with antibiotic treatment, apart fromneutralization of actively secreted bacterial toxins during the activephase of an infection, the liposomal treatment will provide additionalbenefits for a patient by sequestrating the toxins which are releasedduring antibiotic treatment by lysed bacteria, a condition which isknown to be detrimental, for example, during meningitis, inStreptococcus pneumonia infection (acute pneumolysin release) andStreptococcus pyogenes infection (acute release of streptolysin O).

In this combination treatment the empty liposomes and liposome mixturesmay be considered as adjuvants, and the corresponding method oftreatment as adjunct treatment.

A limitation for liposomal therapy is its restricted efficiency inimmunocompromised individuals since the clearance of bacteria lies notwith the chemotherapeutic agent but with the host's own immune system.However, even in immunodeficient patients, liposomal treatment incombination with bactericidal chemotherapy will be beneficial: slowingdown the development of systemic disease, and thus will provide theorganism with the much needed time to allow antibiotics to unfold theirfull bactericidal potential.

Antibiotic treatment considered together with the treatment using emptyliposomes according to the invention is, for example, treatment withcephalosporins and other β-lactam antibiotics, glycopeptides,lincosamides, lipopeptides, macrolides, penicillins and penicillincombinations, quinolones, sulfonamides, chloramphenicol andchloramphenicol analogues, tetracyclines, clindamycin, and folateinhibitors, as listed above. Particular antibiotics considered in atreatment together with empty liposomes of the invention are carbapenemssuch as imipenem, cilastin and meropenem, 2^(nd) generationcephalosporins such as cefuroxime, 3^(rd) generation cephalosporins suchas ceftazidime and ceftriaxone, 4^(th) generation cephalosporins such ascefepime, glycopeptides such as vancomycin, macrolides such asclarithromycin, penicillins such as amoxicillin and flucloxacyllin,penicillin combinations such as amoxicillin/clavulanate andpiperacillin/tazobactam combinations, and quinolones such asciprofloxacin and moxifloxacin, and fluoro-quinolones such aslevofloxacin and gemifloxacin.

All components of the empty liposomes of the invention are substances,which occur naturally in humans. These liposomes are therefore welltolerated and cleared from the body via physiological pathways. Liposomeaerosols should be used for the prevention of pneumonia and otherdiseases of the respiratory tract by the general population duringseasonal influenza epidemics. Most importantly, prophylactic measuresbased on liposome aerosols or other liposome applications will behelpful in the prophylaxis of MRSA pneumonia or bacteremia in hospitals,Pseudomonas aeruginosa, S. aureus or S. pneumonia infections, and inother settings, which favor the spread of infectious diseases.

EXAMPLES Toxins

Streptolysin O (SLO) from Streptococcus pyogenes, α-hemolysin fromStaphylococcus aureus, tetanolysin (TL) from Clostridium tetani andphospholipase C from Clostridium perfringens were purchased from Sigma.Pneumolysin was obtained from Prof. Kadioglu (Cruse G. et al., J.Immunol. 2012; 184:7108-7115). Other toxins include Panton-Valentineleukocidin (PVL) from S. aureus, listeriolysin O (LLO) from Listeriamonocytogenes, perfringolysin O (PFO) from Clostridium perfringens,suilysin (SLY) from S. suis, intermedilysin (ILY) from S. intermedius,cereolysin O (CLO) from B. cereus, thuringiolysin O (TLO) from B.thuringiensis, botulinolysin (BLY) from C. botulinum, sordellilysin(SDL) from C. sordelli, pyolysin (PLO) from Arcanobacterium pyogenes.Culture supernatants from Streptococcus pneumoniae, Streptococcuspyogenes and Staphylococcus aureus were obtained from Profs. K.Mühlemann (Bern) and E. Gulbins (Essen).

Cell Culture

The human embryonic kidney cell line (HEK 293) was maintained asdescribed by Monastyrskaya K et al., Cell Calcium. 2007, 41:207-219. Thehuman acute monocytic leukemia cell line (THP-1) was maintained in RPMI1640 medium containing 10% FBS, 2 mM L-glutamine and 100 U/mlpenicillin, 100 μg/ml streptomycin.

Transfections

CFP (cyan-fluorescent protein) was transiently expressed in HEK 293cells (Monastyrskaya et al., loc. cit.). CFP-expressing HEK 293 cellswere used for laser scanning module (LSM) imaging experiments 2 daysafter transfection.

Liposomes

Cholesterol (Ch) (C-8667), Sphingomyelin (Sm) from chicken egg yolk(S0756), phosphatidylcholine (PC) from soybean (P7443),phosphatidylethanolamine (PE) from bovine brain (P9137) andphospatidylserine (PS) sodium salt from bovine brain (P5660) werepurchased from Sigma. The lipids were individually dissolved inchloroform at 1 mg/ml concentrations and stored at −20° C. For thepreparation of liposomes the chloroform solutions of individual lipidswere mixed in the composition and the proportions, which are given inthe text, to produce routinely 50-500 μl of the final solution.Chloroform was completely evaporated for 20-50 min at 60° C. 50 μl or100 μl of Tyrode's buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mMglucose, 10 mM HEPES; pH=7.4) containing 2.5 mM CaCl₂ was added to thetubes containing films of dried lipids and vigorously vortexed. Thelipid suspensions were incubated for 20-30 min at 45° C. in an Eppendorfthermomixer with vigorous shaking. To produce liposomes, the final lipidsuspensions were sonicated 3×5 sec at 6° C. in a Bandelin Sonopulssonicator at 70% power. The liposomal preparations were left for atleast 1 hour at 6° C. before they were used in experiments. Theconcentration of individual lipids in the liposomes is always given asthe weight per weight (w/w) ratio. In liposomes containing cholesteroland sphingomyelin, the 1:1 (w/w) ratio corresponds to 50% (w/w) or to 66mol % cholesterol. The amounts of liposomes are given as the amount oftotal lipids used for their preparation.

In an alternative method, about 25 ml of each formulation was made bythe ethanol hydration and extrusion method. The final formulations weresterile filtered and filled in autoclave serum glass vials (finalconcentration: 40 mg/ml). The liposome particle sizes are in the rangeof 80-150 nm with good PDI (polydispersity index). The results for theosmolality measurement are also included. They are all in the range ofaround 400 mmol/kg which is pretty close to the desired physiologicallevel.

TABLE 1 Specifications Mean Half- Poly- Zeta diameter width dispersitypotential SD Osmolality Liposomes (nm) (nm) index (mV) (mV) (mmol/kg) pHCh:Sm 130 46 0.13 −1.40 0.6 371 7.02 Sm 81 31 0.15 −3.35 0.5 346 7.02Ch:Sm:PEG2% 116 37 0.11 −9.83 0.7 401 7.03 Ch:Sm:PEG5% 122 43 0.12−15.70 0.5 392 7.04 Sm:PEG2% 96 42 0.19 −9.5 0.5 398 7.02 Sm:PEG5% 11145 0.17 −15.10 0.5 387 7.02

Toxin-Induced Cell Lysis and Protective Effects of Liposomes

In human embryonic kidney epithelial cells (HEK 293), toxin-inducedlysis was monitored as a decline of cytoplasmic fluorescence due to apore-induced efflux of intracellular CFP. Confluent HEK 293 cells seededon 15 mm glass coverslips (2.5×10⁵ cells per coverslip) were mounted ina perfusion chamber at 25° C. in Tyrode's buffer containing 2.5 mM CaCl₂and their fluorescence was recorded in an Axiovert 200 M microscope witha laser scanning module LSM 510 META (Zeiss, Germany) using a ×63 oilimmersion lens (Monastyrskaya et al., loc. cit.). At time-point=0, thebuffer was replaced by 100 μl or 200 μl of the same buffer containingadditionally a cytolytic quantity of a given toxin (e.g. 120 ng of SLOfrom Streptococcus pyogenes) and 20 mM/L dithiotreitol (DTT). Toinvestigate the protective effect of liposomes on toxin-induced celllysis, at time-point=0, cells were routinely challenged with 100 μl of amixture containing toxin/DTT and liposomes of various concentrations andof various lipid composition. The toxin-liposome mixture was preparedimmediately before addition to the cells (with 20 to 30 sec of handlingdelay). In some cases, 100 μl of solution containing liposomes alone wasadded first to the cells followed (with 20 to 30 sec of handling delay)by 100 μl of toxin-containing solution. The protective effects of theliposomes were similar under either experimental condition. The imageswere analyzed using the “Physiology evaluation” software package (Zeiss,Germany).

The effects of purified PFTs or bacterial culture supernatants on theproliferation of a human monocyte cell line (THP-1) were assessed in thepresence or absence of liposomes of various lipid compositions.Routinely, 100-600 μl of toxin-containing solution (Ca²⁺-Tyrode's bufferor BHI broth) was added to 100 μl (5×10⁴ cells) of cells maintained inculture medium and pre-mixed with 50-150 μl of liposomes of variouslipid-composition. After incubation for 3 hours, 1-2 ml of fresh culturemedium was added to the tubes. The cells were counted each day or everysecond day for 8-12 days. The toxins and the liposomes were present forthe whole duration of an experiment. The data presented in the diagramscorrespond to day 5 or day 6, when the cell growth was still in thelinear phase.

Comparison of “Empty” and “Filled” Liposomes

The protection against culture supernatants of S. aureus or S.pneumoniae by the mixture of “empty” Ch:Sm+Sm-only liposomes is comparedwith that of mixture of Ch:Sm+Sm-only liposomes filled with afluorescent dye such as Fluorescein, Oregon Green 488, Rhodamine, orTexas Red.

The protection against culture supernatants of S. aureus or S.pneumoniae by the mixture of “empty” Ch:PC liposomes is compared withthat of mixture of Ch:PC liposomes filled with fluorescent dye such asFluorescein, Oregon Green 488, Rhodamine, or Texas Red.

Protection by Lipid-Coated Surfaces

Beads coated by cholesterol and sphingomyelin are tested for theirtoxin-sequestrating activity against culture supernatants of S. aureusor S. pneumoniae.

In Vivo Effect in Combination with Antibiotic Treatment on BacteremiaInduced by S. aureus or S. pneumoniae.

The 2-component mixture of Ch:Sm and Sm-only liposomes; 3-componentmixture of Ch:Sm; Sm-only and Sm:PC (1:2:2) liposomes and of a4-component mixture of Ch:Sm, Sm-only, Sm:PC and Ch:PC (1:1:1:1) aretested in a mouse models of bacteremia induced by either apenicillin-susceptible strain Streptococcus pneumonia or byMethicillin-resistant Staphylococcus aureus (MSSA). Two types of MSSAstrains are considered, characterized by their ability to secrete or notthe toxin Panton-Valentine leukocidin (PVL). In addition, 2-componentmixture of Ch:Sm and Sm-only pegylated liposomes (2% PEG or 5% PEG) arealso tested.

Laboratory mice are inoculated by intraperitoneal (i.p.), intravenous(i.v.) or intranasal (i.n.) injection of approximately 10⁷ or 10⁸ cfu/mlof bacteria.

For each bacteria strain, each infection route, and for each liposomemixture (LP mixture), intravenous injections of two different doses (2mg/kg or 6 mg/kg) of the LP mixture is started either six hours (t=6),twelve hours (t=12), eighteen hours (t=18), or twenty four hours (t=24)after the bacterial challenge (in each case, the injection of LP mixtureis followed by either one additional injection 12 hours, or twoadditional injections 4 hours and 24 hours after the initial injection),with or without penicillin treatment (30 mg/kg). Antibiotic treatment isinitiated at the same time of liposome treatment. Two types of controlswere performed: infection without treatment and infection treated withantibiotic alone (at t=6, t=12, t=18, or t=24).

For each bacteria strain and for each liposome mixture, and for eachdose of liposome and each route of infection, there were 10 groups ofanimals.

Group 1 No treatment (control) 2 Penicillin alone 3 LP mixture at t = 64 LP mixture at t = 6 + penicillin 5 LP mixture at t = 12 6 LP mixtureat t = 12 + penicillin 7 LP mixture at t = 18 8 LP mixture at t = 18 +penicillin 9 LP mixture at t = 24 10 LP mixture at t = 24 + penicillin

In group 1, the survival of 50% of the group were followed for at least8 days, 25% of the group were euthanized one hour after the bacterialchallenge and the remaining 25% 6 h after the bacterial challenge.

Bacterial counts were determined in blood and several organs such aslung, spleen, and kidney.

Read out: survival, signs of infections, metabolism (serial measurementsof weight loss and recovery, O₂ consumption and CO₂ production ratesmeasured by indirect calorimetry, resting energy expenditure (REE)calculated with the modified Weir formula); inflammation cytokinesprofile (ELISA was performed on serum for tumor necrosis factor(TNF)-alpha, macrophage inflammatory protein (MIP)-2, and IL-1b).

Minimal Bactericidal Concentration (MBC)

No activity of sphingomyelin/cholesterol liposomes against usualstrains:

Strain MBC (mg/mL) ATCC 27853 P. aeruginosa >16 S. aureus >16 762 >16

The testing of the minimum bactericidal concentration (MBC) was carriedout following the guidelines proposed by the Clinical and LaboratoryStandards Institute (CLSI).

For the broth microliter dilution tests, 96-well plates supplementedwith 50 μL of 0.5-16 mg/mL liposomes were inoculated with 50 μL ofMueller Hinton broth containing a bacterial cell suspension of 1-5×10⁵colony-forming units (CFU) per mL of S. aureus. The plates wereincubated for 24 h at 36° C. MBC was determined by transferring 10 μLaliquots from the wells broth microtitre dilution plates onto Columbiablood agar (Oxoid, Wesel, Germany). The inoculated plates were furtherincubated for 24 h at 36° C. and then colonies were counted.

1. Empty liposomes comprising cholesterol and sphingomyelin, andmixtures of empty liposomes comprising cholesterol and sphingomyelin, orphosphatidylcholine and sphingomyelin, with other empty liposomes ofdefined lipid composition for use in the treatment and prevention ofbacterial infections.
 2. Empty liposomes comprising cholesterol andsphingomyelin, and mixtures of empty liposomes comprising cholesteroland sphingomyelin, or phosphatidylcholine and sphingomyelin, with otherempty liposomes comprising lipids or phospholipids selected from thegroup consisting of cholesterol, sphingomyelins, ceramides,phosphatidyl-cholines, phosphatidylethanolamines, phosphatidylserines,diacylglycerols, and phosphatidic acids containing one or two saturatedor unsaturated fatty acids longer than 4 carbon atoms and up to 28carbon atoms, for use in the treatment and prevention of bacterialinfections according to claim
 1. 3. Empty liposomes comprisingsphingomyelin and 30% (w/w) or more cholesterol, and mixtures of emptyliposomes comprising sphingomyelin and 30% (w/w) or more cholesterolwith other empty liposomes of defined lipid composition, for use in thetreatment and prevention of bacterial infections according to claim 1 or2.
 4. Liposome mixture of empty liposomes comprising or consisting ofcholesterol and sphingomyelin, with other empty liposomes comprising orconsisting of sphingomyelin, for use in the treatment and prevention ofbacterial infections according to any one of claims 1 to
 3. 5. Liposomemixture of empty liposomes comprising or consisting ofphosphatidylcholine and sphingomyelin, with other empty liposomescomprising or consisting of sphingomyelin, for use in the treatment andprevention of bacterial infections according claim 1 or
 2. 6. Liposomemixture of empty liposomes comprising or consisting of cholesterol andsphingomyelin with other empty liposomes comprising or consisting ofphosphatidyl-choline and sphingomyelin, and with empty liposomesconsisting of sphingomyelin, for use in the treatment and prevention ofbacterial infections according to any one of claims 1 to
 5. 7. Liposomemixture of empty liposomes comprising or consisting of cholesterol andsphingomyelin with other empty liposomes comprising or consisting ofphosphatidyl-choline and sphingomyelin, with empty liposomes consistingof sphingomyelin, and with empty liposomes comprising or consisting ofcholesterol and phosphatidylcholine, for use in the treatment andprevention of bacterial infections according to any one of claims 1 to6.
 8. Empty liposomes comprising cholesterol and sphingomyelin, andmixtures of empty liposomes comprising cholesterol and sphingomyelin, orphosphatidylcholine and sphingomyelin, with other empty liposomes ofdefined lipid composition, wherein some or all liposomes are modifiedwith polyethylene glycol, for use in the treatment and prevention ofbacterial infections according to any one of claims 1 to
 7. 9. Emptyliposomes comprising cholesterol and sphingomyelin, and mixtures ofempty liposomes comprising cholesterol and sphingomyelin, orphosphatidylcholine and sphingomyelin, with other empty liposomes ofdefined lipid composition, for use in the treatment and prevention ofbacteremia, bacterially infected skin lesions, meningitis andrespiratory tract infections, according to any one of claims 1 to
 8. 10.Lipid bilayers or lipid monolayers comprising cholesterol andsphingomyelin, and optionally phosphatidylcholine, covering non-lipidsurfaces, for use in the treatment and prevention of bacterialinfections.
 11. A mixture of empty liposomes comprising cholesterol andsphingomyelin, or phosphatidylcholine and sphingomyelin, with otherempty liposomes of defined lipid composition.
 12. A mixture according toclaim 11 of empty liposomes comprising cholesterol and sphingomyelin, orphosphatidylcholine and sphingomyelin, with empty liposomes consistingof sphingomyelin.
 13. A mixture according to claim 11 or 12 of emptyliposomes comprising cholesterol and sphingomyelin with other emptyliposomes comprising phosphatidylcholine and sphingomyelin, and withempty liposomes consisting of sphingomyelin.
 14. A mixture according toany one of claims 11 to 13 of empty liposomes comprising cholesterol andsphingomyelin with other empty liposomes comprising phosphatidyl-cholineand sphingomyelin, with empty liposomes consisting of sphingomyelin, andwith empty liposomes comprising cholesterol and phosphatidylcholine. 15.A mixture according to any one of claims 11 to 14 of empty liposomeswherein some or all liposomes are modified with polyethylene glycol. 16.A method of treating bacterial infections comprising administering to apatient in need thereof a therapeutically effective amount of emptyliposomes comprising cholesterol and sphingomyelin, or of a mixture ofempty liposomes comprising cholesterol and sphingomyelin, orphosphatidylcholine and sphingomyelin, with other empty liposomes ofdefined lipid composition.
 17. A method of preventing a bacterialinfection comprising administering to a subject exposed to the risk ofinfection a preventive amount of empty liposomes comprising cholesteroland sphingomyelin, or of a mixture of empty liposomes comprisingcholesterol and sphingomyelin, or phosphatidylcholine and sphingomyelin,with other empty liposomes of defined lipid composition effective forprotection.
 18. A method of treating bacterial infections comprisingadministering to a patient in need thereof a therapeutically effectiveamount of empty liposomes comprising cholesterol and sphingomyelin, orof a mixture of empty liposomes comprising cholesterol andsphingomyelin, or phosphatidylcholine and sphingomyelin, with otherempty liposomes of defined lipid composition before, after, together orin parallel with a standard antibiotic medication against the bacterialinfection.